Lens regeneration is a remarkable phenomenon, which, among vertebrates and during adulthood, occurs only in some urodele amphibians. Upon lentectomy, the pigmented epithelial cells (PECs) from the dorsal iris dedifferentiate and subsequently differentiate into lens cells. While cultured PECs from the ventral iris of the newt or from irises from other animals, including humans, can transdifferentiate to lens, in vivo and especially during adulthood, this ability is restricted to the dorsal iris PECs of a few urodeles only. It has been reasoned, therefore, that these urodeles might hold the key to understanding the mechanisms involved in transdifferentiation with possible applications in other species, especially in humans. We hypothesized previously, (and that hypothesis was the core of our initial proposal), that there must be unique regulatory events in the dorsal iris upon lentectomy. If we could then coax the ventral iris to such regulation we might be able to induce regeneration from the incompetent ventral iris. Our attempts to induce lens regeneration from the ventral iris were eventually successful. Having made such a crucial step, we then performed extensive studies on gene regulation in the ventral and dorsal iris. We have identified very interesting and surprising regulation. In particular, regulatory genes that seemingly are involved in lens regeneration are expressed in both dorsal and ventral irises. Therefore, it seems that both irises initiate the events of regeneration, but somehow there must be a repressive event in the ventral iris. To address this issue we turned our attention to global genomic signatures. We have evidence of two culprits of large-scale regulation. One is differential expression of microRNAs (each one of them is known to regulate hundreds of mRNAs) and the other is specific gene repression-associated histone modifications. Furthermore, our EST and detailed gene expression analysis have revealed that several stem cell-maintaining factors are normally expressed in PECs. This might indicate a relationship of transdifferentiation to stemness and could provided unprecedented insights about the mechanisms of regeneration. To approach these issues we propose to: 1) Analyze the function of miRNAs, which are specifically regulated in the dorsal and ventral iris;2) Analyze the role of histone modifications and the involved enzymes in relation to the process of lens regeneration and 3) correlate the expression of stem cell-maintaining factors to the mechanisms and ability of lens regeneration. PUBLIC HEALTH RELEVANCE: Tissue regeneration could provide solutions to many diseases by repairing the damaged tissue. We are utilizing a vertebrate animal (a salamander), which can regenerate organs and body parts. Our project's subject is regeneration of the eye lens. Successful outcome of our research will have impact in primary and secondary cataracts and other related eye problems. In the long run understanding of the mechanism whereby the salamanders regenerate their tissues will eventually impact the field of regenerative medicine as a whole.